Copper-zinc superoxide dismutase (CuZnSOD) in an enzyme found in relatively high concentrations in the cytoplasm of eukaryotic cells. It consists of two identical subunits, each of which binds one copper (2+) and one zinc (2+) ion in close proximity, held apart by the imidazolate ring of a histidyl residue. CuZnSOD is a very effective catalyst for superoxide disproportionation (2O2- + 2H+ = O2 + H2O2), and has been proposed to act in vivo as a protective agent in the defense system against dioxygen toxicity. Free radicals, including superoxide in particular, have been implicated in carcinogenesis and several other pathological processes as well as in the mechanism of aging. Particular attention has focused recently on the role of free radicals in tissue injury associated with ischemia and subsequent reperfusion of a variety of organs. CuZnSOD is currently being tested for use in treating or preventing free radical- induced injury. CuZnSOD is one of the best characterized non-heme metalloprotein with respect to its physical and spectroscopic properties. It is possible, therefore, to ask unusually detailed questions about chemical properties of CuZnSOD. At the same time, there remain major uncertainties concerning the primary physiological function of CuZnSOD and little information is available concerning any role it may play in metal ion metabolism. The overall goals of this research projects are 1) to study the structure, mechanism, and biological functions of copper-zinc superoxide dismutase by fully characterizing carefully designed derivatives of the protein from Saccharomyces cerevisiae that will be constructed by the techniques of site directed mutagenesis and 2) to investigate general aspects of metal ion- protein interactions using wild type yeast CuZnSOD and genetically modified derivatives as model systems. The mechanistic questions will be addressed by characterizing mutant proteins in which key amino acid residues have been replaced by others with different chemical properties and by examining the effects of making alterations in the metal-binding regions of the protein. Likewise, structural issues and factors involved in stabilizing the protein will be investigated by construction of mutant proteins and by preparation and spectroscopic characterization of isotopically labeled proteins, using strains that overproduce yeast CuZnSOD. The biological consequences of the more interesting mutations will also be examined in vivo after introducing the mutant genes into an CuZnSOD-minus yeast strain. We will also initiate a program of synthesis of new metalloproteins by means of site directed mutagenesis using the yeast CuZnSOD gene as our starting point. The reactivities, physical, and spectroscopic properties of the new proteins will be elucidated. Information gained from this project will add to our understanding of the mechanism of reactions of CuZnSOD and of its physical properties as well as of metalloproteins in general.